The antitumor agents, 6-thioguanine and 6-mercaptopurine, have been reported to produce cellular toxicity as a result of incorporation of either agent into DNA as 6-thioguanine nucleotide. However, there is no data which demonstrates directly that a DNA polymer containing 6-thioguanine has altered functional properties. The purpose of this proposed project will be to ascertain the functional potential of a DNA containing 2-thioguanine in internucleotide linkage, thereby establishing methodology for future studies on the mechanisms of action of other purine and pyrimidine analogs. The three methods of approach will be as follows. I. Allow vaccinia virus to incorporate 6-thioguanine into its DNA, and after harvesting viral particles, determine the ability of the genomes to express their information, namely: 1) ability to induce virus-specific mRNA synthesis and 2) ability to induce the synthesis of virus-specific proteins, i.e., thymidine kinase, DNA polymerase and deoxyribonuclease. The study will be controlled by monitoring the extent of 6-thioguanine incorporation into the viral DNA and relating this level to that found to be toxic to mammalian cells grown in tissue culture. II. The significance of incorporation of thioguanine into cellular DNA on induced-protein synthesis will be evaluated as follows. Cultured cells will be treated with 6-thioguanine and washed free of non-incorporated drug. The cells will then be stimulated with an interferon-inducer or with interferon, and the production of induced interferon and induced antiviral activity by or in the cells will be quantitatively determined. III. The significance of 6-thioguanine metabolites on DNA and protein synthesis in the cell cytoplasm will be determined by pretreating cells with 6-thioguanine. The ability of the pretreated cells to permit vaccinia virus replication will then be assessed. This DNA virus replicates in the cytoplasm independently of the host cell genome.